7-vinylidene cephalosporins and methods of using the same

ABSTRACT

Cephalosporins with an exocyclic allene in the 7-position and their pharmaceutically active salts are potent inhibitors of  beta -lactamases and are therefore useful in the treatment of penicillin resistant infections.

This invention was made with Government support under Contract No.5R01-GM-37774, awarded by the National Institutes of Health. TheGovernment has certain rights in the invention.

This is a Division of application Ser. No. 08/354,858 filed on Dec. 9,1994, now U.S. Pat. No. 5,597,817.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a new class of chemical compounds,specifically 7-vinylidene cephalosporins, pharmaceutically acceptablesalts thereof and a method of inhibiting β-lactamases.

2. Discussion of the Background

The most important mechanism of microbial resistance to β-lactamantibiotics is the bacterial production of β-lactamases, enzymes whichhydrolytically destroy β-lactam antibiotics, such as penicillins andcephalosporins. This type of resistance can be transferred horizontallyby plasmids that are capable of rapidly spreading the resistance, notonly to other members of the same strain, but even to other species. Dueto such rapid gene transfer, a patient can become infected withdifferent organisms, each possessing the same β-lactamase.

β-lactamase enzymes have been organized into four molecular classes: A,B, C, and D based on amino acid sequence. Class A, which includes RTEMand the β-lactamase of Staphylococcus aureus, class C, which includesthe lactamase derived from P99 Enterobacter cloacae, and class D areserine hydrolases. Class A enzymes have a molecular weight of about 29kDa and preferentially hydrolyze penicillins. The class B lactamases aremetalloenzymes and have a broader substrate profile than the proteins inthe other classes. Class C enzymes include the chromosomalcephalosporinases of gram-negative bacteria and have molecular weightsof approximately 39 kDa. The recently recognized class D enzymes exhibita unique substrate profile which differs significantly from both class Aand class C.

The class C cephalosporinases, in particular, are responsible for theresistance of gram-negative bacteria to a variety of both traditionaland newly designed antibiotics. The Enterobacter species, which possessa class C enzyme, are now the third greatest cause of hospital-acquiredinfections in the United States. This class of enzymes often has pooraffinities for inhibitors of the class A enzymes, such as clavulanicacid, a commonly prescribed inhibitor, and to common in vitroinactivators, such as 6-β-iodopenicillanate.

One strategy for overcoming rapidly evolving bacterial resistance is thesynthesis and administration of β-lactamase inhibitors. Frequently,β-lactamase inhibitors do not possess antibiotic activity themselves andare thus administered together with an antibiotic. One example of such asynergistic mixture is "augmentin", which contains the antibioticamoxicillin and the β-lactamase inhibitor, clavulanic acid.

It is thus desirable to find novel β-lactamase inhibitors which can becoadministered with a β-lactam antibiotic.

SUMMARY OF THE INVENTION

Accordingly, one object of the present invention is to provide novelβ-lactamase inhibitors.

It is another object of the present invention to provide pharmaceuticalcompositions useful for inhibiting a β-lactamase.

It is another object of the present invention to provide pharmaceuticalcompositions with increased β-lactam antibiotic activity.

It is another object of the present invention to provide methods ofinhibiting a β-lactamase.

It is another object of the present invention to provide methods ofenhancing the biological activity of a β-lactam antibiotic.

These and other objects, which will become apparent during the followingdetailed description, have been achieved by the inventors' discoverythat compounds of the formula (1) ##STR1## wherein n is 0 or 1 (thesulfide or the sulfone, respectively);

R₁ is selected from the group consisting of

a) hydrogen;

b) linear or branched C₁₋₁₀ -alkyl;

c) halogen;

d) hydroxy-C₁₋₁₀ -alkyl;

e) C₁₋₁₀ -alkoxy;

f) C₂₋₁₀ -alkanoyloxy;

g) C₂₋₁₀ -alkene;

h) C₂₋₁₀ -alkene substituted with one or more groups selected from thegroup consisting of chlorine, fluorine, bromine or phenyl;

i) C₁₋₁₀ -alkoxycarbonyl;

j) C₁₋₁₀ -alkoxycarbamido;

k) N-C₁₋₁₀ -alkoxy-N-C₁₋₁₀ -alkylaminocarbonyl;

l) halo-C₁₋₁₀ -alkyl;

m) C₆₋₁₀ -aryl;

n) C₆₋₁₀ -aryl substituted with one or more groups selected from thegroup consisting of ethyl, n-propyl, isopropyl, amino, methylamino anddimethylamino; and,

o) --COOH or --COOY, wherein Y is a pharmaceutically acceptable cation;

R₂ is selected from the group consisting of

1) --COOH;

2) Cl or F;

3) trifluoromethyl;

4) --CHO; and,

5) --CH₂ M, wherein M is selected from the group consisting of

a) hydrogen;

b) halo;

c) hydroxy;

d) C₁₋₁₀ -alkoxy;

e) C₆₋₁₀ -aryloxy;

f) C₆₋₁₀ -aryl-C₁₋₁₀ -alkoxy;

g) mercapto;

h) mercapto substituted with one or more groups selected from the groupconsisting of methyl, ethyl or phenyl;

i) C₂₋₁₀ -acylthio;

j) C₂₋₁₀ -acyloxy or carbamoyloxy;

k) C₂₋₁₀ -acyloxy or carbamoyloxy substituted with one or more groupsselected from the group consisting of --COOH, aminophenyl, phenyl, C₁₋₆-alkyl, chlorine, bromine or fluorine;

l) a quaternary ammonium salt;

m) amino or amido; and,

n) amino or amido substituted with one or more groups selected from thegroup consisting of C₁₋₁₀ -alkyl groups;

R₃ is selected from the group consisting of

a) hydrogen; and,

b) pharmaceutically acceptable cations;

are effective β-lactamase inhibitors.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Thus, in a first embodiment, the present invention provides novelcompounds of the formula (1) ##STR2## wherein n is 0 or 1;

R₁ is selected from the group consisting of

a) hydrogen;

b) linear or branched C₁₋₁₀ -alkyl, preferably, C₁₋₆ -alkyl, morepreferably, methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl,n-pentyl, cyclopropyl, cyclopentyl, or cyclohexyl, most preferablyt-butyl;

c) halogen, preferably Br or Cl;

d) hydroxy-C₁₋₁₀ -alkyl, preferably, hydroxy-C₁₋₆ -alkyl, morepreferably, hydroxymethyl, 1-hydroxyethyl or 2-hydroxyethyl;

e) C₁₋₁₀ -alkoxy, preferably, C₁₋₆ -alkoxy, more preferably, t-butoxy ormethoxy;

f) C₂₋₁₀ -alkanoyloxy, preferably, C₂₋₆ -alkanoyloxy, more preferably,acetoxy or propanoyloxy;

g) C₂₋₁₀ -alkene, preferably, C₂₋₆ -alkene, more preferably, ethylene,1-propylene or 2-propylene;

h) substituted C₂₋₁₀ -alkene, preferably, C₂₋₆ -alkene, more preferablyethylene, 1-propylene or 2-propylene, wherein said substituents are onemore groups selected from the group consisting of chlorine, fluorine,bromine or phenyl;

i) C₁₋₁₀ -alkoxycarbonyl, preferably, C₁₋₆ -alkoxycarbonyl, morepreferably, methoxycarbonyl or t-butoxycarbonyl;

j) C₁₋₁₀ -alkoxycarbamido, preferably, C₁₋₆ -alkoxycarbamido, morepreferably, methoxycarbamido, ethoxycarbamido or n-propoxycarbamido;

k) N-C₁₋₁₀ -alkoxy-N-C₁₋₁₀ -alkylaminocarbonyl, preferably, N-C₁₋₆-alkoxy-N-C₁₋₆ -alkylaminocarbonyl, more preferably,N-methoxy-N-methylaminocarbonyl, N-ethoxy-N-methylaminocarbonyl,N-methoxy-N-ethylaminocarbonyl or N-ethoxy-N-ethylaminocarbonyl;

l) halo-C₁₋₁₀ -alkyl, preferably, halo-C₁₋₆ -alkyl, more preferably,chloromethyl, 1-chloroethyl or 2-chloroethyl;

m) C₆₋₁₀ -aryl group, preferably, phenyl, tolyl, anisoyl, mesityl, andxylyl;

n) substituted C₁₋₁₀ -alkyl, preferably, phenyl, tolyl, anisoyl,mesityl, and xylyl, wherein said substituents are one or more groupsselected from the group consisting of ethyl, n-propyl, isopropyl, amino,methylamino and dimethylamino; and,

o) --COOH or --COOY, wherein Y is a pharmaceutically acceptable cation,preferably, sodium, potassium, calcium or any other pharmaceuticallyacceptable cation known in the art; and,

R₂ is selected from the group consisting of

1) --COOH;

2) Cl or F;

3) trifluoromethyl;

4) --CHO; and,

5) --CH₂ M, wherein M is selected from the group consisting of

a) hydrogen;

b) halo, preferably F, Cl, Br, or I;

c) hydroxy;

d) C₁₋₁₀ -alkoxy, preferably, C₁₋₆ -alkoxy, more preferably, methoxy,ethoxy, n-propoxy or isopropoxy;

e) C₆₋₁₀ -aryloxy, preferably, C₆₋₁₀ -aryloxy, more preferably, phenoxyor naphthoxy;

f) C₆₋₁₀ -aryl-C₁₋₁₀ -alkoxy, preferably, C₆₋₁₀ -aryl-C₁₋₆ -alkoxy, morepreferably, phenylmethoxy, 1-phenylethoxy or 2-phenylethoxy;

g) mercapto, preferably, thiol;

h) substituted mercapto, preferably, thiol, wherein said substituentsare selected from the group consisting of methyl, ethyl or phenyl;

i) C₂₋₁₀ -acylthio, preferably C₂₋₆ -acylthio, more preferably,acetylthio or propanoylthio;

j) C₂₋₁₀ -acyloxy or carbamoyloxy, preferably, C₂₋₆ -alkanoyloxy, C₆₋₁₀-aryl-carbonyloxy, carbamoyloxy or thiocarbamoyloxy, more preferably,acetoxy or benzoyloxy;

k) substituted C₂₋₁₀ -acyloxy or carbamoyloxy, preferably, C₂₋₆-alkanoyloxy, C₆₋₁₀ -aryl-carbonyloxy, N-C₁₋₆ -alkylcarbamoyloxy,N,N-di-C₁₋₆ -alkylcarbamoyloxy, thiocarbamoyloxy, N-C₁₋₆-alkylthiocarbamoyloxy or N,N-di-C₁₋₆ -alkylthiocarbamoyloxy, morepreferably, acetoxy, α-aminophenylacetoxy, benzoyloxy,benzyloxycarbonyloxy, succinoyloxy, N-methylcarbamoyloxy,N-ethylcarbamoyloxy, N,N-dimethylcarbamoyloxy, N,N-diethylcarbamoyloxy,N-methylthiocarbamoyloxy, N-ethylthiocarbamoyloxy,N,N-dimethylthiocarbamoyloxy, N,N-diethylthiocarbamoyloxy, wherein saidsubstituents are one or more groups selected from the group consistingof --COOH, aminophenyl, phenyl, methyl, ethyl, chlorine, bromine orfluorine;

l) a quaternary ammonium salt, preferably trimethyl ammonium chloride ortriethyl ammonium chloride;

m) amino or amido group, preferably --NH₂ or --CONH₂ ; and,

n) substituted amino or amido group, preferably --NH₂ or --CONH₂,wherein said substituents are one or two C₁₋₁₀ -alkyl groups, preferablyC₁₋₆ -alkyl groups, more preferably, methyl, ethyl, n-propyl, isopropylor n-butyl;

R₃ is selected from the group consisting of

a) hydrogen; and,

b) pharmaceutically acceptable cations, preferably, sodium, potassium orcalcium.

In a preferred embodiment, n is 0 and R₁ is selected from the groupconsisting of t-butyl, bromine and hydrogen.

In a more preferred embodiment, n is 0, R₁ is selected from the groupconsisting of t-butyl, bromine and hydrogen and R₂ is --CH₂ OAc.

In another preferred embodiment, n is 1 and R₁ is selected from thegroup consisting of t-butyl, bromine and hydrogen.

In another more preferred embodiment, n is 1, R₁ is selected from thegroup consisting of t-butyl, bromine and hydrogen and R₂ is --CH₂ OAc.

In a most preferred embodiment, n is 1, R₁ is t-butyl, R₂ is --CH₂ OAcand R₃ is sodium.

The compounds of this invention are generally prepared from7-aminocephalosporanic acid. Their preparation is as follows. Chiral7-aminocephalosporanic acid, 2, was esterified with diphenyldiazomethaneto obtain 3. Treatment of 3 with excess triethylamine andtrifluoromethanesulfonic anhydride, followed by hydrolysis of theresultant trifluorosulfonyl imine produced benzhydryl7-oxocephalosporanate 4. Due to its instability, this ketone was usedwithout further purification. The reaction between 4 andethynylmagnesium bromide stereospecifically produced the correspondingpropargylic alcohol 5. Conversion to the triflate 6 withtrifluoromethanesulfonic anhydride, followed by careful treatment witheither (t-C₄ H₉)₂ CuCNLi₂ or with copper(I) bromide, yielded eitherbenzhydryl 7-(2'α-t-butylvinylidene) cephalosporanate 7 or benzhydryl7-(2'α-bromovinylidene) cephalosporanate 8, respectively. The synthesisof these chiral allenes proceeded with 100% stereospecificity via ananti S_(n) 2' displacement of the leaving group. Reduction of 8 with theZn-Cu couple produced the parent terminally unsubstituted benzhydryl7-vinylidene cephalosporanate 9, as shown below. ##STR3##

Deprotection of compounds 7, 8, and 9 produced the corresponding sodiumsalts 13, 14, and 15, respectively. Sulfones 10, 11, and 12 weresynthesized by oxidation of 7, 8, and 9 with excess m-chloroperbenzoicacid (m-CPBA). Sodium salts 16 and 17 could be obtained by thedeprotection of sulfones 10 and 12, respectively. The reaction scheme isshown below. ##STR4##

Compounds having R₁ groups other than those shown above may be made byforming a vinylidene anion and reacting it with an electrophile. Thevinylidene anion may be made by a standard lithium-halogen (ormagnesium-halogen) exchange reaction, for example, reaction of 14 withmethyl lithium. The lithium vinylidene may then be functionalized byreaction with an electrophile, for example, an alkoxycarbonyl chloride,a N-alkoxy-N-alkylaminocarbonyl chloride, a dihaloalkyl, an epoxide (toform a hydroxy-alkyl), or carbon dioxide.

The compounds wherein R₁ is alkoxy, alkanoyloxy, alkene, alkoxycarbamidoor aryl may be made by a nucleophilic attack of an appropriate anion(i.e., alkoxide, alkanoyloxide, alkenyl anion, alkyoxycarbamidyl anion,or aryl anion) on the propargylic triflate 6. Thus, sodium methoxide maybe used to add to compound 6 to form the 7-methoxy-vinylidene.

The compounds wherein R₂ is a halogen may be formed by displacement ofthe --OAc group with ethylxanthate (EtOCS₂ K). Raney-Nickeldesulfurization (H₂ /Ra-Ni) would yield the exocyclic alkene which maythen be ozonized to the 3-hydroxy cephem. Reaction with a halogenatingreagent would provide the 3-halo species. For example, PCl₅ may be usedto convert the 3-OH group into a 3-Cl group. The 3-methyl species may beobtained by the rearrangement of the exocyclic alkene, formed byRaney-Nickel desulfurization, by reaction with Et₃ N. The3-hydroxymethyl species may be obtained by hydrolysis of the --OAc groupwith NaOH or an appropriate enzyme. The 3-halomethyl species may beformed by reaction of the 3-hydroxymethyl species with a halogenatingreagent. For example, PCl₅ may be used to form the 3-chloromethylspecies.

The compounds wherein M is alkoxy, aryloxy, or arylalkoxy may beobtained by reaction of the 3-hydroxymethyl species with tosyl chlorideand displacement of the resultant tosylate with an oxide. For example,sodium methoxide may be used to obtain the 3-methoxymethyl species. Thecompounds wherein M is mercapto may be formed by reaction of the3-chloromethyl compound with sodium sulfhydride (NaSH). This compoundmay be further derivatized with an alkylhalide to form a substitutedmercapto or an acylchloride to form an acylthio group.

The species wherein M is an amino group may be formed by the GabrielSynthesis, i.e., reaction of the 3-chloromethyl compound with potassiumphthalimide and hydrolysis of the product with acid to yield the3-aminomethyl compound. The 3-ammoniomethyl compound may be formed byreaction of the 3-aminomethyl compound with methyl chloride to form the3-trimethylammoniomethyl chloride.

The compound wherein M is an amido group (CONH₂) may be formed bydisplacement of the tosylate described above with cyanide, e.g., KCN,followed by hydrolysis of the resulting nitrile to the amide.

The activity of the present compounds against β-lactamases wasdetermined by measuring the IC₅₀. The IC₅₀ value represents theconcentration of inhibitor required to effect a 50% loss of activity offree enzyme. The present 7-vinylidenecephems were evaluated asinhibitors of the β-lactamase of Enterobacter cloacae P99. The IC₅₀value of each compound was determined as follows. Following a 10 minuteincubation of a dilute solution of enzyme (2.56 nM) and inhibitor (<0.64mM), a 50 mL aliquot of this incubation mixture was then further dilutedinto 1 mL nitrocefin solution, and the rate of hydrolysis was measuredduring a 1 minute period by monitoring the absorbance of nitrocefin as afunction of time. The present 7-vinylidenecephems were evaluatedrelative to known inhibitors tazobactam, clavulanic acid and6-(2'α-t-butylvinylidene) penam sulfone. The results are summarized inTable 1 below.

                  TABLE 1                                                         ______________________________________                                        P99 β-lactamase inhibitory activity                                                                  IC.sub.50                                         Compound                    (mg/mL)                                           ______________________________________                                        7-(2'α-t-butylvinylidene) cephem (13)                                                               3.91                                              7-(2'α-t-butylvinylidene) cephem sulfone(16)                                                        0.05                                              7-(2'α-t-butyl-2'β-deuteriovinylidene) cephem sulfone                                          0.10                                              7-vinylidenecephem (15)     25.83                                             7-vinylidenecephem sulfone (17)                                                                           0.11                                              6-(2'α-t-butylvinylidene) penam sulfone                                                             0.39                                              7-(2'α-bromovinylidene) cephem (14)                                                                 6.16                                              Tazobactam                  0.22                                              Clavulanic acid             17.24                                             ______________________________________                                    

The compounds of the formula I are surprisingly potent inhibitors ofβ-lactamase, particularly β-lactamase of the class C. All of compoundsmade according to this formula are found to have IC₅₀ values competitivewith commercially available clavulanic acid, and several of thecompounds are competitive with commercially available tazobactam.Compound (16) 7-(2'α-t-butylvinylidene)cephem sulfone was found to bethe most potent inhibitor, approximately five-fold better thantazobactam and 350-fold better than clavulanic acid. Thus the compoundsof the formula I are useful as β-lactamase inhibitors.

In a second embodiment, the present invention provides pharmaceuticalcompositions useful for inhibiting a β-lactamase. The presentpharmaceutical compositions comprise at least one of the present7-vinylidene cephalosporins and at least one pharmaceutically acceptablecarrier.

The present compositions may be employed in capsule form or as tablets,powders or liquid solutions or as suspensions or elixirs. They may beadministered orally, intravenously or intramuscularly. The presentcompositions are preferably presented in a form suitable for absorptionby the gastrointestinal tract.

Tablets and capsules for oral administration may be in unit dosepresentation form, and may contain conventional pharmaceutical carrierssuch as binding agents, for example, syrup, acacia, gelatin, sorbitol,tragacanth, or polyvinylpyrrolidone; fillers, for example, lactose,sugar, maize-starch, calcium phosphate, sorbitol or glycine, lubricants,for example, magnesium stearate, talc, polyethylene glycol, silica;disintegrants, for example, potato starch; or acceptable wetting agentssuch as sodium lauryl sulphate. The tablets may be coated according tomethods well known in the art. Oral liquid preparations may be in theform of aqueous or oily suspensions, solutions, emulsions, syrups,elixirs, etc. or may be presented as a dry product, for reconstructionwith water or other suitable vehicle before use. Such liquidpreparations may contain conventional additives such as suspendingagents, for example, sorbitol syrup, methyl cellulose, glucose/sugarsyrup, gelatin, hydroxyethylcellulose, carboxymethyl cellulose, aluminumstearate gel or hydrogenated edible fats; emulsifying agents, forexample lecithin, sorbitan monooleate or acacia; non-aqueous vehicles,which may include edible oils, for example, almond oil, fractionatedcoconut oil, oily esters, propylene glycol, or ethyl alcohol;preservatives, for example, methyl or propyl p-hydroxybenzoates orsorbic acid. Suppositories will contain conventional suppository bases,e.g., cocoa butter or other glyceride.

Compositions for injection may be presented in unit does form inampules, or in multidose containers with an added preservative. Thecompositions may take such forms as suspensions, solutions, emulsions inoily or aqueous vehicles, and may contain formulatory agents such assuspending, stabilizing and/or dispersing agents. Alternatively, theactive ingredient may be in powder form for reconstitution with asuitable vehicle, e.g., sterile, pyrogen-free water, before use.

The present compositions may also be prepared in suitable forms forabsorption through the mucous membranes of the nose and throat orbronchial tissues and may conveniently take the form of powder or liquidsprays or inhalants, lozenges, throat paints, etc. For medication of theeyes or ears, the preparations may be presented as individual capsules,in liquid or semi-solid form, or may be used as drops, etc. Topicalapplications may be formulated in hydrophobic or hydrophilic bases asointments, creams, lotions, paints, powders, etc.

Also, in addition to a carrier, the present compositions may includeother ingredients such as stabilizers, binders, antioxidants,preservatives, lubricators, suspending agents, viscosity agents orflavoring agents and the like.

For veterinary medicine, the composition may, for example, be formulatedas an intramammary preparation in either long acting or quick-releasebases.

The dosage to be administered depends to a large extent upon thecondition of the subject being treated and the weight of the subject,the route and frequency of administration, the parenteral route beingpreferred for generalized infections and the oral route for intestinalinfections.

The instant compositions may be administered in several unit dosageforms as, for example, in solid or liquid orally ingestible dosage form.The compositions per unit dosage, whether liquid or solid may containfrom 0.1% to 99% of active material (the present 7-vinylidenecephalosporins and optional antibiotic), the preferred range being fromabout 10-60%. The composition will generally contain from about 15 mg toabout 1500 mg by weight of active ingredient based upon the total weightof the composition; however, in general, it is preferable to employ adosage amount in the range of from about 250 mg to 1000 mg. Inparenteral administration the unit dosage is usually the pure compoundin a slightly acidified sterile water solution or in the form of asoluble powder intended for solution.

The present β-lactamase inhibitors will be particularly useful in thetreatment of infections caused by Enterobacter, Citrobacter, andSerratia. These bacteria have the ability to attach to the epithelialcells of the bladder or kidney (causing urinary tract infections) andare resistant to multiple antibiotics including amoxicillin andampicillin. The present β-lactamase inhibitors would also be useful inthe treatment of infections caused by highly resistant Pneumococci. Suchdiseases include otitis media, sinusitis, meningitis (both in childrenand adults), bacteremia, and septic arthritis. Resistant pneumococcalstrains have surfaced in many parts of the world. For example, inHungary, 58% of S. pneumoniae are resistant to penicillin, and 70% ofchildren who are colonized with S. pneumonniae carry resistant strainsthat are also resistant to tetracycline, erythromycin,trimethoprin/sulfamethoxazole (TMP/SMX), and 30% resistant tochloroanphenicol. Klebsiella pneumoniae (resistant via the production ofβ-lactamase) have caused hospital outbreaks of wound infection andsepticemia.

Thus, in a third embodiment, the present invention providespharmaceutical compositions with increased β-lactam antibiotic activity.This pharmaceutical composition is as defined above, but in addition toat least one of the present 7-vinylidene cephalosporins and at least onea pharmaceutically acceptable carrier, the compositions also contains atleast one β-lactam antibiotic. The β-lactam antibiotic may be any of theabove-noted antibiotics or any other known in the art, preferablyamoxicillin or piperacillin, and its selection will depend upon whatindication is necessary.

In a fourth embodiment, the present invention provides a method ofinhibiting a β-lactamase, comprising administering to a patient in needthereof an effective amount of at least one of the present 7-vinylidenecephalosporins. The method of administration may be any of theabove-noted methods or any other known to one of skill in the art.

In a fifth embodiment, the present invention provides a method ofenhancing the biological activity of a β-lactam antibiotic bycoadministering to a patient in need thereof, an effective amount of oneof the present 7-vinylidene cephalosporins and an effective amount of atleast one β-lactam antibiotic. The method of administration may be anyof the above-noted methods or any other known to one of skill in theart. The β-lactam antibiotic may be any of the above-noted β-lactamantibiotics or any other known in the art.

Other features of the invention will become apparent in the course ofthe following descriptions of exemplary embodiments which are given forillustration of the invention and are not intended to be limitingthereof.

EXAMPLES EXAMPLE 1 Benzhydryl 7β-Aminocephalosporanate (3)

The title compound was prepared according to the procedure of Sheehanand Commons. To a suspension of 7-aminocephalosporanic acid, 2, (130.4g, 0.48 mol) in methanol (480 mL) was added a solution ofdiphenyldiazomethane (93.0 g, 0.48 mol) in CH₂ Cl₂. The suspension wasstirred (mechanically) at room temperature for 44 hours. The solid wasremoved by filtration, the organic layer was concentrated in vacuo andpurified by column chromatography (10% CH₃ OH in CH₂ Cl₂) to afford thedesired ester as a pale yellow solid (86.1 g, 41% yield). R_(f) =0.44 in1:9 CH₃ OH:CH₂ Cl₂ ; mp 45°-46° C.; IR (CHCl₃) 2980, 1780, 1730 cm⁻¹ ; ¹H NMR (CDCl₃) δ 8.41 (2H, bs), 7.22 (10H, m), 6.91 (1H, s), 5.27 (1H, d,J=2.8 Hz), 5.15 (1H, d, A of ABq, J=14.0 Hz), 4.94 (1H, s), 4.84 (1H, d,B of ABq, J=14.0 Hz), 3.73 (1H, d, A of ABq, J=16.7 Hz), 3.33 (1H, d, Bof ABq, J=16.7 Hz), 1.92 (3H, s); ¹³ C NMR (CDCl₃) δ 169.8, 168.8,160.6, 138.9, 138.7, 129.5, 129.3, 129.1, 128.7, 128.5, 127.97, 127.61,127.52, 127.18, 126.52, 126.06, 125.4, 79.0, 63.3, 62.6, 58.5, 25.7,20.1.

EXAMPLE 2 Benzhydryl 7-Oxocephalosporanate

The title compound was prepared by modifying the procedure of Hagiwara,D. F.; Sawada, K.; Ohnami, T.; Aratani, M.; Hashimoto, M. J. Chem Soc.Chem. Commun. 1982, 578. To a solution of benzhydryl7β-aminocephalosporanate, 3, (5.9 g, 13.5 mmol) in anhydrous CH₂ Cl₂ (70mL) at -78° C., triethylamine (5.6 mL, 40.4 mmol) was added dropwisewith stirring. After 5 minutes, trifluoromethane-sulfonic anhydride (6.8mL, 40.4 mmol) was added dropwise to this solution over a 5 minuteperiod. The reaction mixture was allowed to warm slowly to 0° C. over a1 hour period. It was then cooled to -78° C. and triethylamine (5.6 mL,40.4 mmol) was added over approximately 5 minutes. The reaction mixturewas stirred at -78° C. for an additional 30 minutes, followed by theaddition of 0.5N HCl (50 mL). The cooling bath was removed and theresultant solution was further stirred for 30 minutes. The layers wereseparated and the aqueous layer was extracted with CH₂ Cl₂ (50 mL). Thecombined organic layers were washed with cold phosphate buffer, pH 5.7(5×200 mL), dried (Na₂ SO₄), and concentrated to produce the titlecompound (5.8 g, 98% yield) as a pale yellow solid which was usedwithout further purification. IR (CHCl₃) 3005, 1830, 1790, 1740 cm⁻¹ ; ¹H NMR (CDCl₃) δ 7.39 (10H, m), 7.05 (1H, s), 5.07 (1H, d, A of ABq,J=13.9 Hz), 4.85 (1H, d, B of ABq, J=14.0 Hz), 3.64 (1H, d, A of ABq,J=18.5 Hz), 3.44 (1H, d, B of ABq, J=18.6 Hz), 2.05 (3H, s); ¹³ C NMR(CDCl₃) δ 188.4 (s), 170.3 (s), 160.1 (s), 158.7 (s), 138.8 (s), 138.6(s), 128.4, 128.2, 128.1, 127.7, 126.9, 126.2, 80.1 (d), 65.8 (d), 62.6(t), 27.7 (t), 20.4 (q).

EXAMPLE 3 Benzhydryl 7α-Ethynyl-7β-hydroxycephalosporanate (5)

Ethynylmagnesium bromide (45.2 mL, 22.6 mmol) was slowly added to a cold(-78° C.) solution of 7-oxocephalosporanate, 4, (5.5 g, 12.6 mmol) inanhydrous THF (85 mL). It was then stirred at -78° C. for 1 hour and at-40° C. for 1.5 hours. The reaction mixture was quenched with aceticacid (2.9 mL, 50.4 mmol) and ether (500 mL) was added. The combinedorganic layers were washed with water (1×100 mL), brine (1×100 mL),dried (Na₂ SO₄), and concentrated in vacuo. The product was immediatelypurified by column chromatography (1:4 EtOAc:CH₂ Cl₂) to give the titlecompound (2.9 g, 50% yield) as pale yellowish fluffy solid. R_(f) =0.56in 1:4 EtOAc:CH₂ Cl₂ ; mp 50°-52° C.; IR (CHCl₃) 3670, 3565, 3300, 3010,2120, 1790, 1730 cm⁻¹ ; ¹ H NMR (CDCl₃) δ 7.37 (10H, m), 6.95 (1H, s),5.14 (1H, d, A of ABq, J=13.9 Hz), 5.08 (1H, s), 4.89 (1H, d, B of ABq,J=13.9 Hz), 3.53 (1H, d, A of ABq, J=17.8 Hz), 3.35 (1H, d, B of ABq,J=17.8 Hz), 2.88 (1H, s), 2.05 (3H, s); ¹³ C NMR (CDCl₃) δ 170.7 (s),162.7 (s), 160.3 (s), 139.3 (s), 139.1 (s), 132.1 (s), 128.42, 128.0,127.3, 126.9, 125.6, 79.6 (d), 78.3 (s), 77.9 (s), 77.3 (d), 65.4 (d),62.6 (t), 26.3 (t), 20.5 (q). Anal. Calcd for C₂₅ H₂₁ NO₆ S: C, 64.79;H, 4.54; N, 3.02. Found: C, 64.20; H, 4.39; N, 3.25.

EXAMPLE 4 Benzhydryl 7α-Ethynyl-7β-trifluoromethanesulfonatoCephalosporanate (6)

Trifluoromethanesulfonic anhydride (3.3 mL, 19.1 mmol) was addeddropwise (4 s intervals) to a cold (0° C.) solution of pyridine (2.6 mL,31.8 mmol) and benzhydryl 7α-ethynyl-7β-hydroxycephalosporanate, 5, (5.9g, 12.7 mmol) in anhydrous CH₂ Cl₂ (60 mL). The reaction mixture wasallowed to warm to room temperature and monitored by TLC (reactiontime=30 minutes). After concentration the residue was purified by columnchromatography (CH₂ Cl₂) to yield the title compound as a white solid(4.67 g, 62% yield). R_(f) =0.63 in 15% EtOAc in CH₂ Cl₂ ; mp 42°-43°C.; IR (CHCl₃) 3300, 3020, 2120, 1810, 1780, 1750 cm⁻¹ ; ¹ H NMR (CDCl₃)δ 7.39 (10H, m), 6.94 (1H, s), 5.29 (1H, d, A of ABq, J=13.9 Hz), 5.26(1H, s), 5.09 (1H, d, B of ABq, J=14.8 Hz), 3.52 (1H, d, A of ABq,J=16.5 Hz), 3.34 (1H, d, B of ABq, J=18.3 Hz), 3.29 (1H, s), 2.09 (3H,s); ¹³ C NMR (CDCl₃) δ 170.1 (s), 159.4 (s), 155.1 (s), 140.8 (s), 139.2(s), 139.1 (s), 128.5, 128.1, 126.9, 126.8, 125.3, 118.0 (q, J=321.11Hz), 87.3 (s), 84.0 (d), 79.6 (d), 71.9 (s), 66.5 (d), 61.7 (t), 26.5(t), 20.4 (q). Anal. Calcd for C₂₆ H₂₀ NO₈ S₂ F₃ : C, 52.44; H, 3.36; N,2.35; F, 9.58. Found: C, 52.66; H, 3.37; N, 2.33; F, 9.26.

EXAMPLE 5 Benzhydryl 7-(2'α-bromovinylidene) Cephalosporanate (8)

Method A: Copper (I) bromide (CuBr, 133 mg, 0.93 mmol) was added in oneportion to a solution of benzhydryl7α-ethynyl-7β-(trifluoromethanesulfonato) cephalosporanate, 6, (500 mg,0.84 mmol) in anhydrous DMF (5.0 mL) at room temperature and stirred inthe dark for 30 minutes. The DMF was removed in vacuo at roomtemperature. The residue was dissolved in ether (50 mL), washed withwater (2×15 mL), dried (Na₂ SO₄), and concentrated to produce a yellowsolid. This material was purified by column chromatography (CH₂ Cl₂) toyield the title compound as a pale yellow solid (140 mg, 32% yield).R_(f) =0.75 in 15% EtOAc in CH₂ Cl₂ ; mp 63°-65° C.; IR (CHCl₃) 3010,1950, 1780, 1730 cm⁻¹ ; ¹ H NMR (CDCl₃) δ 7.42 (10H, m), 7.01 (1H, s),6.74 (1H, d, J=1.17 Hz), 5.38 (1H, d, J=1.12 Hz), 5.02 (1H, d, A of ABq,J=13.5 Hz), 4.78 (1H, d, B of ABq, J=13.4 Hz), 3.60 (1H, d, A of ABq,J=18.31 Hz), 3.41 (1H, d, B of ABq, J=18.11 Hz), 2.04 (3H, s); ¹³ C NMR(CDCl₃) δ 194.6 (s), 170.3 (s), 160.6 (s), 156.1 (s), 139.1 (s), 138.9(s), 128.4, 128.1, 128.0, 127.7, 127.1, 124.6, 111.7 (s), 81.8 (d), 79.9(d), 62.9 (t), 56.2 (d), 27.8 (t), 20.5 (q); HRMS calcd for C₂₅ H₂₀ NO₅SBrNa!⁺, i.e. M+Na!⁺, m/z calcd 548.0143, found 548.0146.

Method B: Lithium bromide (LiBr, 285 mg, 3.3 mmol), and copper (I)bromide (CuBr, 470 mg, 3.3 mmol) were added in one portion to a solutionof benzhydryl 6α-ethynyl-6β-(trifluoromethanesulfonato)cephalosporanate, 6, (1.5 g, 2.5 mmol) in anhydrous THF (15 mL). Themixture was allowed to stir at room temperature for 5 minutes. The THFwas removed in vacuo. The residue was dissolved in ether (20 mL), washedwith water (1×10 mL), dried (Na₂ SO₄), and concentrated in vacuo to giveyellow solid (1.30 g, 98% yield). This reaction produced a single isomer7-(2'α-bromovinylidene) cephalosporanate. The material was judged to be95% pure by ¹ H NMR and used for next step without purification.

EXAMPLE 6 Benzhydryl 7-Vinylidenecephalosporanate (9)

To a solution of benzhydryl 7-(2'α-bromovinylidene) cephalosporanate, 8,(2.4 g, 4.6 mmol) in a 1:5 mixture of anhydrous THF:MeOH (60 ml) wasadded NH₄ Cl (0.98 g, 18.4 mmol) and Zn-Cu couple (0.6 g, 9.2 mmol).After stirring at room temperature for 30 minutes, the reaction mixturewas concentrated in vacuo. The residue was dissolved in ether (100 mL),washed with water (20 mL), dried (Na₂ SO₄), concentrated, andchromatographed (1:1 Hexane:CH₂ Cl₂ followed by 1:3 Hexane:CH₂ Cl₂) togive a white fluffy solid (1.45 g, 71% yield). R_(f) =0.3 in CH₂ Cl2; IR(CHCl₃) 3010, 1985, 1790, 1730 cm⁻¹ ; ¹ H NMR (CDCl₃) δ 7.40 (10H, m),7.0 (1H, s), 5.58 (2H, d, J=13.4 Hz), 5.29 (1H, t, J=1.88 Hz), 4.99 (1H,d, A of ABq, J=13.35 Hz), 4.74 (1H, d, B of ABq, J=13.3 Hz), 3.57 (1H,d, A of ABq, J=18.2 Hz), 3.37 (1H, d, B of ABq, J=18.3 Hz), 2.03 (3H,s); ¹³ C NMR (CDCl₃) δ 200.0 (s), 170.3 (s), 160.8 (s), 158.7 (s), 139.2(s), 139.0 (s), 128.4, 128.0, 127.9, 127.7, 127.4, 127.0, 123.0, 105.7(s), 85.1 (t), 79.7 (d), 63.0 (t), 56.6 (d), 27.8 (t), 20.5 (q); HRMScalcd for C₂₄ H₂₁ NO₅ SNa!⁺, i.e. M+Na!⁺, m/z calcd 470.1038, found470.1042.

EXAMPLE 7 Benzhydryl 7-(2'α-t-Buytlvinylidene) Cephalosporanate (7)

To a suspension of CuCN (0.376 g, 4.2 mmol) in anhydrous THF (30 mL) wasadded t-BuLi (4.0 mL, 1.7M in pentane, 6.8 mmol) at -100° C. The coolingbath was removed until all the solid had gone into the solution(approximately 3 minutes). This solution was again cooled to -100° C.and was cannulated into a cold (-100° C.) solution of benzhydryl7α-ethynyl-7β-(trifluoromethanesulfonato) cephalosporanate, 6, (2.0 g,3.4 mmol) in anhydrous THF (5 mL) at -100° C. The solution was furtherstirred at -100° C. for 1 minute before pouring the cold reactionmixture into cold (0° C.) saturated NH₄ Cl solution (100 mL). Thereaction mixture was extracted with ether (2×50 mL), dried (Na₂ SO₄),concentrated, and chromatographed (5% EtOAc in CH₂ Cl₂) to give a whitefluffy solid (0.913 g, 54% yield). R_(f) =0.80 in 5% EtOAc in CH₂ Cl₂ ;mp 113°-114° C.; IR (CHCl₃) 3000, 2960, 1970, 1770, 1730 cm⁻¹ ; ¹ H NMR(CDCl₃) δ 7.42 (10H, m), 7.05 (1H, s), 5.98 (1H, d, J=1.63 Hz), 5.25(1H, d, J=1.69 Hz), 4.97 (1H, d, A of ABq, J=13.30 Hz), 4.72 (1H, d, Bof ABq, J=13.23 Hz), 3.55 (1H, d, A of ABq, J=18.14 Hz), 3.35 (1H, d, Bof ABq, J=18.23 Hz), 2.01 (3H, s), 1.18 (9H, s); ¹³ C NMR (CDCl₃) δ194.6 (s), 170.2 (s), 161.1 (s), 159.5 (s), 139.2 (s), 139.0 (s),128.3,128.0,127.9, 127.7, 127.1, 121.9, 113.2 (d), 107.2 (s), 79.6 (d),63.0 (t), 57.0 (d), 33.6 (s), 29.7 (q), 27.8 (t), 20.5 (q). Anal. Calcdfor C₂₉ H₂₉ NO₅ S: C, 69.18; H, 5.77; N, 2.78. Found: C, 69.08; H, 6.00;N, 2.73.

EXAMPLE 8 Benzhydryl 7-(2'α-Bromovinylidene) Cephalosporanate Sulfone(11)

To the solution of sulfide 8 (0.65 g, 1.23 mmol) in CH₂ Cl₂ (10 mL) andpH=6.4 phosphate buffer solution (10 mL), was added in one portionm-CPBA (85%, 0.853 g, 4.94 mmol). The mixture was stirred as rapidly aspossible overnight at room temperature. After separating the layers, theaqueous layer was extracted with ether (1×10 mL). The combined organiclayers were washed with 5% NaHSO₃ (1×5 mL), saturated NaHCO₃ (1×5 mL),dried (Na₂ SO₄), concentrated, and chromatographed 5% EtOAc in CH₂ Cl₂.There was obtained a yellow solid (0.32 g, 46% yield). R_(f) =0.35 in 5%EtOAc in CH₂ Cl₂ ; IR (CHCl₃) 3020, 1950, 1800, 1740, 1350, 1130 cm⁻¹ ;¹ H NMR (CDCl₃) δ 7.39 (10H, m), 7.00 (1H, s), 6.94 (1H, d, J=1.38 Hz),5.39 (1H, d, J=1.13 Hz), 5.09 (1H, d, A of ABq, J=14.25 Hz), 4.75 (1H,d, B of ABq, J=14.33 Hz), 4.06 (1H, d, A of ABq, J=18.25 Hz), 3.82 (1H,d, B of ABq, J=18.17 Hz), 2.06 (3H, s); ¹³ C NMR (CDCl₃) δ 196.8, 170.1,159.6, 155.3, 138.7, 138.6, 128.6, 128.4, 128.3, 127.6, 127.1, 125.8,125.4, 104.4, 83.8, 80.6, 69.0, 61.9, 51.3, 20.4.

EXAMPLE 9 Benzhydryl 7-Vinylidene Cephalosporanate Sulfone (12)

This compound was prepared from the sulfide 9 as described for thebenzhydryl 7-(2'α-bromovinylidene) cephalosporanate sulfone (11)(Example 8) (yield=55%, 0.590 g). R_(f) =0.35 in 5% EtOAc in CH₂ Cl₂ ;mp 155°-156° C.; IR (CHCl₃) 3010, 1985, 1790, 1730, 1340, 1125 cm⁻¹ ; ¹H NMR (CDCl₃) δ 7.43 (10H, m), 6.99 (1H, s), 5.70 (2H, dd, J=1.65 Hz,J=5.31 Hz), 5.33 (1H, s), 5.03 (1H, d, A of ABq, J=14.02 Hz), 4.70 (1H,d, B of ABq, J=14.01 Hz), 4.04 (1H, d, A of ABq, J=8.12 Hz), 3.79 (1H,d, B of ABq, J=18.40 Hz), 2.03 (3H, s); ¹³ C NMR (CDCl₃) δ 201.7 (s),170.1 (s), 159.8 (s), 157.6 (s), 138.8 (s), 138.6 (s), 128.4, 128.2,128.1, 127.5, 127.0, 124.0, 98.8 (s), 86.4 (t), 80.3 (d), 69.5 (d), 61.9(t), 51.0 (t), 20.3 (q); HRMS calcd for C₂₅ H₂₁ NO₇ SNa!⁺, i.e. M+Na!⁺,m/z calcd 502.0936, found 502.0931.

EXAMPLE 10 Benzhydryl 7-(2'α-t-butylvinylidene) Cephalosporanate Sulfone(10)

This compound was prepared from the sulfide 7 as described above inbenzhydryl 7-(2'α-bromovinylidene) cephalosporanate sulfone (11)(Example 8) (yield=65%, 0.692 g). R_(f) =0.42 in 2% EtOAc in CH₂ Cl₂ ;mp 163°-164° C.; IR (CHCl₃) 3010, 2960, 1970, 1790, 1740, 1340, 1125cm⁻¹ ; ¹ H NMR (CDCl₃) δ 7.40 (10H, m), 7.01 (1H, s), 6.18 (1H, d,J=1.66 Hz), 5.30 (1H, s), 5.02 (1H, d, A of ABq, J=13.93 Hz), 4.68 (1H,d, B of ABq, J=13.93 Hz), 4.02 (1H, d, A of ABq, J=18.27 Hz), 3.76 (1H,d, B of ABq, J=18.20 Hz), 2.03 (3H, s), 1.19 (9H, s); ¹³ C NMR (CDCl₃) δ197.1 (s), 170.1 (s), 160.0 (s), 158.6 (s), 138.8 (s), 138.7 (s), 128.5,128.3, 128.2, 127.6, 127.1, 126.5, 123.1, 114.6 (d), 100.1 (s), 80.4(d), 70.1 (d), 62.0 (t), 51.2 (t), 34.0 (s), 29.7 (q), 20.4 (q); HRMScalcd for C₂₉ H₂₉ NO₇ SNa!⁺, i.e. M+Na!⁺, m/z calcd 558.1562, found558.1567.

EXAMPLE 11 Sodium Salt of 7-(2'α-Bromovinylidene) Cephalosporanic Acid(14)

To a solution of benzhydryl 7-(2'α-bromovinylidene)-cephalosporanate,11, (300 mg, 0.57 mmol), in anhydrous CH₂ Cl₂ (6 mL) was added anisole(0.62 mL, 5.7 mmol) at -78° C. followed by addition of AlCl₃ solution(1.43 mL, 1.0M in nitrobenzene, 1.43 mmol) in one portion. The mixturewas stirred for 15 minutes at -78° C. and poured into rapidly stirredcold water (60 mL) containing NaHCO₃ (0.48 g, 5.7 mmol) followed byaddition of EtOAc (50 mL). It was further stirred for 5 minutes andfiltered using celite 545. The aqueous layer was separated andconcentrated in vacuum to 2 mL and further purified by reverse phasepreparative layer chromatography (R_(f) =0.62 in 5% EtOH in water).Lyopholization produced a pale yellow fluffy solid (105 mg, 48% yield).IR (nujol) 1975, 1750, 1610, 1410 cm⁻¹ ; ¹ H NMR (d⁶ -DMSO) δ 7.56 (1H,d, J=0.7 Hz), 5.62 (1H, s), 5.00 (1H, d, A of ABq, J=13.5 Hz), 4.76 (1H,d, B of ABq, J=13.28 Hz), 3.56 (1H, d, A of ABq, J=17.91 Hz), 3.17 (1H,d, B of ABq, J=17.84 Hz), 2.05 (3H, s); HRMS calcd for C₁₂ H₉ NO₅ SBrNa₂!⁺, i.e. M+Na!⁺, m/z calcd 403.9180, found 403.9168.

EXAMPLE 12 Sodium Salt of 7-Vinylidene Cephalosporanic Acid (15)

This compound was prepared from the corresponding ester 9 (500 mg, 1.12mmol) as described for the sodium salt of 7-(2'α-bromovinylidene)cephalosporanic acid (14) above (Example 11) to give the title compoundas a white fluffy solid (220 mg, 65% yield). R_(f) =0.65 in 5% EtOH inwater. IR (nujol) 1980, 1760, 1735, 1590 cm⁻¹ ; ¹ H NMR (d₆ -DMSO) δ5.81 (2H, d, J=1.5 Hz), 5.41 (1H, s), 4.95 (1H, d, A of ABq, J=12.11Hz), 4.71 (1H, d, B of ABq, J=12.0 5 Hz), 3.50 (1H, d, A of ABq, J=17.20Hz), 3.24 (1H, d, B of ABq, J=17.40 Hz), 1.99 (3H, s); HRMS calcd forC₁₂ H₁₁ NO₅ SNa!⁺, i.e. M+H!⁺, m/z calcd 304.0255, found 304.0250.

EXAMPLE 13 Sodium Salt of 7-(2'α-t-Butylvinylidene) Cephalosporanic Acid(13)

This compound was prepared from the corresponding ester 7 (420 mg, 0.83mmol) as described in sodium salt of 7-(2'α-bromovinylidene)cephalosporanic acid (14) above (Example 11) to give title compound as awhite fluffy solid (118 mg, 39% yield). R_(f) =0.80 in 15% EtOH inwater. IR (nujol) 1975, 1760, 1720, 1610 cm⁻¹ ; ¹ H NMR (d₆ -DMSO) δ6.20 (1H, s), 5.33 (1H, s), 4.93 (1H, d, A of ABq, J=12.02 Hz), 4.69(1H, d, B of ABq, J=11.97 Hz), 3.48 (1H, d, A of ABq, J=17.66 Hz), 3.20(1H, d, B of ABq, J=18.09 Hz), 1.99 (3H, s), 1.10 (9H, s); HRMS calcdfor C₁₆ H₁₉ NO₅ SNa!⁺, i.e. M+H!⁺, m/z calcd 360.0881, found 360.0882.

EXAMPLE 14 Sodium Salt of 7-(2'α-t-Butylvinylidene) Cephalosporanic AcidSulfone (16)

This compound was prepared from the corresponding ester 10 (250 mg, 0.47mmol) as described in sodium salt of 7-(2'α-bromovinylidene)cephalosporanic acid (14) (Example 11) above to give the title compoundas a white fluffy solid (110 mg, 60% yield). R_(f) =0.50 in 20% EtOH inwater; IR (nujol) 1980, 1765, 1730, 1615, 1330, 1130 cm⁻¹. ¹ H NMR (d₆-DMSO) δ 6.39 (1H, s), 5.84 (1H, s), 4.92 (1H, d, A of ABq, J=12.07 Hz),4.64 (1H, d, B of ABq, J=12.08 Hz), 4.05 (1H, d, A of ABq, J=17.71 Hz),3.78 (1H, d, B of ABq, J=17.63 Hz), 1.99 (3H, s), 1.10 (9H, s); HRMScalcd for C₁₆ H₁₉ NO₇ SNa!⁺, i.e. M+H!⁺, m/z calcd 392.0779, found392.0780.

Obviously, numerous modifications and variations of the presentinvention are possible in light of the above teachings. It is,therefore, to be understood that within the scope of the appendedclaims, the invention may be practiced otherwise than as specificallydescribed herein.

What is claimed as new and desired to be secured by Letters Patent ofthe United States is:
 1. A method of enhancing the biological activityof a β-lactam antibiotic, comprising:coadministering to a patient inneed thereof an effective amount of a compound of the formula (1)##STR5## wherein n is 0 or 1; R₁ is selected from the group consistingofa) hydrogen; b) linear or branched C₁₋₁₀ -alkyl; c) halogen; d)hydroxy-C₁₋₁₀ -alkyl; e) C₁₋₁₀ -alkoxy; f) C₂₋₁₀ -alkanoyloxy; g) C₂₋₁₀-alkene; h) C₂₋₁₀ -alkene substituted with one or more groups selectedfrom the group consisting of chlorine, fluorine, bromine or phenyl; i)C₁₋₁₀ -alkoxycarbonyl; j) C₁₋₁₀ -alkoxycarbamido; k) N-C₁₋₁₀-alkoxy-N-C₁₋₁₀ -alkylaminocarbonyl; l) halo-C₁₋₁₀ -alkyl; m) C₆₋₁₀-aryl; n) C₆₋₁₀ -aryl substituted with one or more groups selected fromthe group consisting of ethyl, n-propyl, isopropyl, amino, methylaminoand dimethylamino; o) --COOH or --COOY, wherein Y is a pharmaceuticallyacceptable cation; R₂ is selected from the group consisting of1) --COOH;2) Cl or F; 3) trifluoromethyl; 4) --CHO; and, 5) --CH₂ M, wherein M isselected from the group consisting ofa) hydrogen; b) halo; c) hydroxy;d) C₁₋₁₀ -alkoxy; e) C₆₋₁₀ -aryloxy; f) C₆₋₁₀ -aryl-C₁₋₁₀ -alkoxy; g)mercapto; h) mercapto substituted with one or more groups selected fromthe group consisting of methyl, ethyl or phenyl; i) C₂₋₁₀ -acylthio; j)C₂₋₁₀ -acyloxy or carbamoyloxy; k) C₂₋₁₀ -acyloxy or carbamoyloxysubstituted with one or more groups selected from the group consistingof --COOH, aminophenyl, phenyl, C₁₋₆ -alkyl, chlorine, bromine orfluorine; l) a quaternary ammonium salt; m) amino or amido group; and n)substituted amino or amido group; R₃ is selected from the groupconsisting ofa) hydrogen; and, b) pharmaceutically acceptable cations;andan effective amount of a β-lactam antibiotic.
 2. The method of claim1 wherein the β-lactam antibiotic is amoxicillin or piperacillin.
 3. Apharmaceutical composition, comprising: an effective amount of acompound of the formula (1) ##STR6## wherein n is 0 or 1; R₁ is selectedfrom the group consisting ofa) hydrogen; b) linear or branched C₁₋₁₀-alkyl; c) halogen; d) hydroxy-C₁₋₁₀ -alkyl; e) C₁₋₁₀ -alkoxy; f) C₂₋₁₀-alkanoyloxy; g) C₂₋₁₀ -alkene; h) C₂₋₁₀ -alkene substituted with one ormore groups selected from the group consisting of chlorine, fluorine,bromine or phenyl; i) C₁₋₁₀ -alkoxycarbonyl; j) C₁₋₁₀ -alkoxycarbamido;k) N-C₁₋₁₀ -alkoxy-N-C₁₋₁₀ -alkylaminocarbonyl; l) halo-C₁₋₁₀ -alkyl; m)C₆₋₁₀ -aryl; n) C₆₋₁₀ -aryl substituted with one or more groups selectedfrom the group consisting of ethyl, n-propyl, isopropyl, amino,methylamino and dimethylamino; o) --COOH or --COOY, wherein Y is apharmaceutically acceptable cation; R₂ is selected from the groupconsisting of1) --COOH; 2) Cl or F; 3) trifluoromethyl; 4) --CHO; and,5) --CH₂ M, wherein M is selected from the group consisting ofa)hydrogen; b) halo; c) hydroxy; d) C₁₋₁₀ -alkoxy; e) C₆₋₁₀ -aryloxy; f)C₆₋₁₀ -aryl-C₁₋₁₀ -alkoxy; g) mercapto; h) mercapto substituted with oneor more groups selected from the group consisting of methyl, ethyl orphenyl; i) C₂₋₁₀ -acylthio; j) C₂₋₁₀ -acyloxy or carbamoyloxy; k) C₂₋₁₀-acyloxy or carbamoyloxy substituted with one or more groups selectedfrom the group consisting of --COOH, aminophenyl, phenyl, C₁₋₆ -alkyl,chlorine, bromine or fluorine; l) a quaternary ammonium salt; m) aminoor amido group; and n) substituted amino or amido group; R₃ is selectedfrom the group consisting ofa) hydrogen; and, b) pharmaceuticallyacceptable cations; an effective amount ofa β-lactam antibiotic; and apharmaceutically acceptable carrier.
 4. The composition according toclaim 3, wherein the β-lactam antibiotic is amoxicillin or piperacillin.